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J. Trop. Resour. Sustain. Sci. 3 (2015): 72-76 Organic Acid Content and Antimicrobial Properties of Eleiodoxa conferta Extracts at Different Maturity Stages Seri Intan Mokhtar*, Nur Ain Abd Aziz Faculty of Agro Based Industry, Universiti Malaysia Kelantan, Jeli Campus, Locked Bag No.100, 17600 Jeli, Kelantan, Malaysia. Ab st ract Available online 4 May 2015 Keywords: Eleiodoxa conferta water extracts at different maturity stages were shown to Eleiodoxa conferta, fruit maturity, contain three types of organic acid which are oxalic, ascorbic and malic acids antimicrobial, organic acid. by HPLC analysis. The content of oxalic acid concentration was the highest at -1 -1 young stage (1.33 gml ) followed by mature stage (1.26 gml ) and ripe stage at -1 -1 ⌧*Corresponding author: 1.23 gml . Malic acid content decreased during ripening from 1.38 gml to 1.07 Assoc. Prof. Dr, Seri Intan gml-1. The concentration of ascorbic acid remained constants during the fruit Mokhtar, ripening. When antimicrobial activity of the extract was tested against several Faculty of Agro Based Industry, Universiti Malaysia Kelantan, Jeli bacteria it was observed that the activity decreased as the fruit ripen. Highest Campus, Locked Bag No.100, diameter of inhibition zone was recorded against Escherichia coli by the young 17600 Jeli, Kelantan, Malaysia. fruit extract at 15.3 mm. MIC of 0.063 gml-1 exhibit by the young fruit extract Email: [email protected] was helpful in controlling the growth of Gram negative bacteria, E. coli. However, 0.063 gml-1 concentration of extract from mature fruit is shown to regulate the growth of Gram positive bacteria, Staphylococus aureus. © 2015 UMK Publisher. All rights reserved. 1. Introduction longkong (Aglaia dookoo Griff.) at ripe stage and Eleiodoxa conferta commonly called kelubi showed that citric acid and malic acid were present at or asam paya belongs to the family Arecaceae is an the level of 0.22 % w/w and 0.15% w/w. [3] reported underutilized fruit that has not been reported about their citric acid as the major organic acid in fully ripened antimicrobial activity and organic acids content. Found strawberry and mulberry genotype fruit. [4] reported in peat swamp and fresh water swamps area. It is that malic acid is the dominant acid in various types of clustering palm which forms dense thickets [1]. The olive fruit such as Memecik, Domat and Akhisar Uslu leaves are up to 3.5 metres long, arranged along the varieties in ripe stage. This result is contrast to the rachis and toothed along the margins. Fruits skin has recent studied by [2] reported that organic acids in scaly appearance and very sour taste, reddish colour foreign fruit such as strawberry, sweet cherry, and when ripe and has one or occasionally two seeds which mulberry increased as maturity progressed. [5] can eat fresh or pickles for sale in local markets. identified three organic acids in orange juice and wines: Organic acids are great significance in plants. citric, ascorbic and malic acids. The major organic acid −1 As intermediates in the metabolic processes of the was citric acid in orange juice (12.66 g L ) and wine −1 fruits including respiration and photosynthesis, these (6.03 g L ). acids are contributed in growth, maturation and In recent years, researchers have found that senescence. [2] studied the organic acid in tropical fruit organic acids in some fruits can inhibit growth of some bacteria and fungi. [6] discovered that malic acid could ISSN Number: 2289-3946 © 2015 UMK Publisher. All rights reserved. 72 J. Trop. Resour. Sustain. Sci. 3 (2015): 72-76 inhibit the growth of Listeria monocytogenes, aureus and Bacillus cereus), three Gram-negative Salmonella gaminara, and Escherichia coli. [7] also (Pseudomonas aeruginosa, Escherichia coli and found that organic acids such as malic, citric, lactic, and Salmonella spp.) were obtained from Director of tartaric acid had antibacterial activity with specific pH Veterinary Research Institute, Ipoh, Malaysia. The test conditions. bacteria was sub-cultured into nutrient agar [10] and In view of these opinions and observations, maintained at 4°C respectively. All the bacteria were the present investigation was taken up to screen this sub-cultured on nutrient agar for inoculum with a loop. underutilized fruit to investigate its organic acids Single colony of each subculture bacteria was content and antimicrobial activity at different maturity inoculated in 10 ml LB broth and incubated at 37°C for stages. 24 hours on incubator shaker at 150 rpm. These stock cultures were kept at 4°C for antimicrobial assays [11]. 2. Materials and Methods The disc diffusion test was performed using a modified Kirby-Bauer method [12]. 100 µl of the microbial 2.1. Organic acid content analysis Fruit of Eleiodoxa conferta was collected suspension was added and swabbed onto the surface of from local fruit farm in Jeli, Kelantan. The fruit was Nutrient Agar (NA). The plates were allowed to dry at identified by Dr Shamsul Mohamad, lecturer in room temperature within 15 minutes. Next, 10 µl of Universiti Malaysia Kelantan. Fruits with good extract were immersed into a sterile 6 mm paper discs condition at each maturity stages were selected. The and allowed to dry for 1 hour in a laminar flow hood. ripening stage of the fruits was distinguished through Dry discs were aseptically placed on NA. The plates physical observation. To obtain fruits extract, water were incubated at 37°C for 24 h. Antimicrobial activity extraction method was applied. Fruit was cut into two was evaluated by measuring inhibition zone diameters. halves, and 50 g fruit was extracted by addition of 100 The experiment was done in triplicate. The minimum ml of water using electric blender. To obtain clear juice, inhibitory concentrations (MICs) of all fruit extracts the fruits extract was filtered through a muslin cloth or against 5 microbial strains were determined using a a stainless steel filter with small porosity. The filtered broth dilution method. The fruit extract was diluted juices were immediately stored in freezer at 0°C for using serial two fold dilution to obtain 7 concentrations further analysis and also to avoid any colour changes. (0.5 mgml-1 to 0.008 mgml-1) [13]. Then 1 ml of the To start the analysis, the fruits extracts were clarified microbial suspension of Staphylococcus aureus and Escherichia coli (2 X 106 cfuml-1) were added to each by centrifugation at 5000 rpm for 10 minutes and filtered through a 0.45 µm filter membrane before test tube and mixed thoroughly [14]. The control test injection [8]. Two ml of extract was transferred into tube containing the medium only was used to confirm HPLC vial. HPLC analysis was carried out using the sterility of the medium. Then the culture tubes were Shimadzu HPLC equipped with a column oven C18 incubated at 37°C for 24 hours. Afterwards, incubation used for separation of organic acids. The mobile phase tubes were observed for changes in turbidity as an employed was ammonium di-hydrogen phosphate indicator of growth. The lowest concentration that did buffer (NH4H2PO4, 0.5% w/v). The flow rate of the not permit any visible growth was considered as MIC. mobile phase was maintained at 0.5 mlmin-1, the injection volume was 1 µL and the temperature of 2.3. Statistical analysis column oven was set at 40°C [9]. The detection Experimental data were analysed using wavelength was 254 nm. Organic acids were identified analysis of variance (ANOVA) and significant and quantified by comparison of their retention time differences among means at p < 0.05 were determined and peak area to the standard. by Tukey-test using MINITAB statistical software (Version 16). 2.2. Antimicrobial properties Five species of bacteria were used in this study consists of two Gram-positive (Staphylococcus ISSN Number: 2289-3946 © 2015 UMK Publisher. All rights reserved. 73 J. Trop. Resour. Sustain. Sci. 3 (2015): 72-76 3. Results and Discussion 3.2. Screening for antimicrobial activity at 3.1. The composition and content of organic different maturity stages of fruit extracts acids at different maturity stages of Young stage of extracts exhibited good activity against all the bacteria species as shown in extracts As shown in the Fig. 1, there were significant Table 1. Highest diameter of inhibition zone was differences in the contents of organic acid among recorded against Escherichia coli by the young furit different maturity stages of extracts except for ascorbic extact at 15.3 mm. Water extract of Eleiodoxa conferta acid. The main organic acid in Eleiodoxa conferta was exhibited good activity (diameter zone of inhibition > oxalic acid. The content of oxalic acid concentration 8 mm) at the order of Salmonella spp > Pseudomonas was the highest at young stage [1.33 gml-1] followed aeruginosa for Gram negative bacteria and Bacillus by mature stage (1.26 gml-1) and ripe stage of 1.23 cereus > Staphylococcus aureus for Gram positive gml-1. Malic acid content decreased during ripening bacteria. from 1.38 gml-1 to 1.07 gml-1. The concentration of ascorbic acid remained constants during the fruit ripening. Oxalic acid Malic acid Ascorbic acid 1.6 a ) a 1 1.4 - b ab a b 1.2 a 1 0.8 0.6 0.4 Concentration (gml Concentration 0.2 a a a 0 Young Mature Ripe Maturity stages Figure 1: Distribution of organic acids at different maturity stages of Eleiodoxa conferta extracts at three different maturity stages. Different letters (a-c) in each stage of fruits indicate significant differences at p < 0.05. Each point represented the mean and error bars were ± SD of the mean. Table 2: Zone of inhibition of Eleiodoxa conferta extracts at different maturity stages tested against Gram positive and Gram Negative bacteria using disc diffusion method Zone of inhibition [mm] Stage of fruit Gram +ve bacteria Gram -ve bacteria development SA BC EC PA SS Young 10.7 13.3 15.3 9.3 10.7 Mature 10.7 11.3 12.7 8.7 10.3 Ripe 12.3 12 10.7 8.7 8.3 SA – Staphylococcus aureus, BC – Bacillus cereus, EC – Escherichia coli, PA – Pseudomonas aeruginosa, SS – Salmonella spp.

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